scholarly journals Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Feixiang Wu ◽  
Yantao Liu ◽  
Xin Lv ◽  
Xuerong Miao ◽  
Yuming Sun ◽  
...  
Keyword(s):  
Lung Injury ◽  
Critical Role ◽  
Saline Control ◽  
Control Group ◽  
Tlr4 Expression ◽  
Small Interference Rna ◽  
Tlr4 Gene ◽  
Interference Rna

Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA) targeting Toll-like receptor 4 (TLR4) gene in ameliorating lipopolysaccharide- (LPS-) induced acute lung injury (ALI).Methods.In vitro, alveolar macrophages (AMs) were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL) for 2 h, and the function and expression of TLR4 were evaluated.In vivo, rats received intratracheal injection of 300 μL of normal saline (control group), 300 μL of Ad-EGFP (Ad-EGFP group), or 300 μL of Ad-siTLR4 (Ad-siTLR4 group) and then were intravenously treated with LPS (50 mg/kg) to induce ALI.Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment bothin vitroandin vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals.Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expressionin vitroandin vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

Tat-BMPs-PAMAM Conjugates Enhance Therapeutic Effect of Small Interference RNA on U251 Glioma Cells In Vitro and In Vivo

2010 ◽  
Vol 21 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Lei Han ◽  
Anling Zhang ◽  
Hanjie Wang ◽  
Peiyu Pu ◽  
Xinguo Jiang ◽  
...  
Keyword(s):  
Therapeutic Effect ◽  
Glioma Cells ◽  
Small Interference Rna ◽  
Interference Rna

Sphingosine kinase 1 regulates lysyl oxidase through STAT3 in hyperoxia-mediated neonatal lung injury

Thorax ◽  
2021 ◽  
pp. thoraxjnl-2020-216469
Author(s):  
Alison W Ha ◽  
Tao Bai ◽  
David L Ebenezer ◽  
Tanvi Sethi ◽  
Tara Sudhadevi ◽  
...  
Keyword(s):  
Lung Injury ◽  
Lysyl Oxidase ◽  
Critical Role ◽  
Sphingosine Kinase ◽  
In Vivo Studies ◽  
Sphingosine Kinase 1 ◽  
Neonatal Lung ◽  
Neonatal Lung Injury

IntroductionNeonatal lung injury as a consequence of hyperoxia (HO) therapy and ventilator care contribute to the development of bronchopulmonary dysplasia (BPD). Increased expression and activity of lysyl oxidase (LOX), a key enzyme that cross-links collagen, was associated with increased sphingosine kinase 1 (SPHK1) in human BPD. We, therefore, examined closely the link between LOX and SPHK1 in BPD.MethodThe enzyme expression of SPHK1 and LOX were assessed in lung tissues of human BPD using immunohistochemistry and quantified (Halo). In vivo studies were based on Sphk1−/− and matched wild type (WT) neonatal mice exposed to HO while treated with PF543, an inhibitor of SPHK1. In vitro mechanistic studies used human lung microvascular endothelial cells (HLMVECs).ResultsBoth SPHK1 and LOX expressions were increased in lungs of patients with BPD. Tracheal aspirates from patients with BPD had increased LOX, correlating with sphingosine-1-phosphate (S1P) levels. HO-induced increase of LOX in lungs were attenuated in both Sphk1−/− and PF543-treated WT mice, accompanied by reduced collagen staining (sirius red). PF543 reduced LOX activity in both bronchoalveolar lavage fluid and supernatant of HLMVECs following HO. In silico analysis revealed STAT3 as a potential transcriptional regulator of LOX. In HLMVECs, following HO, ChIP assay confirmed increased STAT3 binding to LOX promoter. SPHK1 inhibition reduced phosphorylation of STAT3. Antibody to S1P and siRNA against SPNS2, S1P receptor 1 (S1P1) and STAT3 reduced LOX expression.ConclusionHO-induced SPHK1/S1P signalling axis plays a critical role in transcriptional regulation of LOX expression via SPNS2, S1P1 and STAT3 in lung endothelium.

314 MELATONIN ON IN VIVO AND IN VITRO MATURATION OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 246 ◽  
Author(s):  
H. Fernandes ◽  
L. Schefer ◽  
F. C. Castro ◽  
C. L. V. Leal
Keyword(s):  
Ovarian Stimulation ◽  
In Vitro Maturation ◽  
F1 Hybrids ◽  
Saline Control ◽  
Control Group ◽  
Mouse Oocytes ◽  
Maturation Rate ◽  
Different Doses

Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation of oocytes: COC were collected 48 h after 5 IU of eCG and maturated in the presence of 3 different doses of melatonin (10–9, 10–6, and 10–3 M) or 0.5 µg mL–1 of FSH (control) for 16 h. In vitro-maturing oocytes were in incubated at 37°C, 5% CO2, and 95% humidity. Maturation rates were evaluated according to the presence of the first polar body under an inverted microscope. Statistical analyses were performed by ANOVA followed by Tukey's test (4 replicates). In the first treatment, 20 mg kg–1 of melatonin showed the highest in vivo maturation rate, 80.3% (61/76), while 10 mg kg–1 of melatonin was 62.4% (53/85) and the saline control group was 69.4% (77/111), but differences were not significant (P > 0.05). For in vitro maturation of oocytes from animals previously treated with melatonin, the 10 mg kg–1 of melatonin group had the highest maturation rate, 53.2% (99/186), in comparison with the saline and 20 mg kg–1 of melatonin groups, which showed 46.6 (88/189) and 39.0% (85/218), respectively; again, no differences were detected (P > 0.05). In the last treatment, the maturation rates increased from 48.9 (43/88) to 53.7 (51/95) and 56.0% (56/100) as the melatonin concentrations decreased from 10–3, 10–6, and 10–9 M, respectively. The control group had the highest rate of 57.3% (55/96), but no statistical differences were observed (P = 0.706). In conclusion, under the conditions studied, melatonin was unable to improve the maturation rate neither after in vivo nor in vitro treatment. However, during in vitro maturation, melatonin alone was as efficient as FSH in promoting maturation in murine oocytes, indicating its potential effect on stimulating meiosis. Therefore, the role of melatonin in stimulating meiosis needs further investigation.Acknowledgments to FAPESP for fellowship (HF) and funding (CLVL).

Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Silvia I García ◽  
Ludmila S Peres Diaz ◽  
Maia Aisicovich ◽  
Mariano L Schuman ◽  
María S Landa
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Cardiac Hypertrophy ◽  
Structural Changes ◽  
In Vitro Models ◽  
Heart Weight ◽  
Saline Control ◽  
Control Group

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

Small interference RNA targeting Krüppel-like factor 8 inhibits the renal carcinoma 786-0 cells growth in vitro and in vivo

2010 ◽  
Vol 136 (8) ◽  
pp. 1255-1265 ◽  
Author(s):  
Wei-Jin Fu ◽  
Jia-Chu Li ◽  
Xiao-yun Wu ◽  
Zhan-Bing Yang ◽  
Zeng-Nan Mo ◽  
...  
Keyword(s):  
Renal Carcinoma ◽  
Small Interference Rna ◽  
Rna Targeting ◽  
Interference Rna

Overcoming Drug Resistance In Acute Lymphoblastic Leukemia by Inhibition of CBP/γ-Catenin

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3264-3264
Author(s):  
Enzi Jiang ◽  
Eugene Park ◽  
Cu Nguyen ◽  
James Yoon ◽  
Yao-Te Hsieh ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Western Blot ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Saline Control ◽  
Control Group ◽  
Apoptosis Protein

Abstract Abstract 3264 Survivin, an inhibitor of apoptosis protein (IAP) family, has been associated with poor prognosis in cancer including leukemia. Survivin can be downregulated in colon cancer cells by inhibition of the β-catenin/Creb-binding protein (CBP) interaction using ICG-001, a small molecule specific inhibitor of the β-catenin/CBP interaction. We have shown previously that combined ICG-001 and chemotherapy can downregulate Survivin and sensitize ALL cells to chemotherapy in vitro and in a pilot study in vivo. In this study, we determine the CBP interaction with ICG-001 in primary ALL cells and preclinically evaluate ICG-001 in vitro and in vivo as an adjuvant against primary ALL and. For this purpose, primary ALL cells were co-cultured with OP9 cells and treated for 4 days with ICG-001 (10mM, 20mM) or DMSO as vehicle control. Mean viability (trypan blue exclusion) of cells treated with ICG-001 was significantly lower (ICG-001 10mM: 75.12% ± 3.15%; 20mM: 41.18%± 7.88%) compared to cells treated with DMSO (84.99% ± 0.42%) (% cell viability relative to initial control) (p=0.03). Real time RT-PCR showed ICG-001 dose-dependent downregulation of Survivin in ALL compared to control (ICG10mM vs. control: p=0.0037 and 20mM vs. control: p=0.0031). Immunoblotting demonstrated reduction of Survivin after ICG-001 treatment. Primary ALL cells incubated with a combination of VDL (Vincristine, Dexamethasone and L-Asparaginase) and ICG-001 showed decreased viability (28.7%± 4.9%) versus VDL only (79.3%± 13.6%) (p=0.014) determined by MTT assay. To elucidate if ICG-001 interacts with β-catenin/CBP as shown previously in colon cancer, we analyzed ten primary pre-B ALL cells and found significantly greater γ-catenin and Survivin expression versus normal pre-B-Cells. β-catenin was absent or in some cases expressed only weakly. Expression of v-catenin and b-catenin in ALL xenograft cells were detected by Western blot. One primary ALL was selected and incubated with γ-catenin and β-catenin siRNA for 48hrs, followed by 6hrs incubation with Wnt3a. Wnt3a induced both of γ-catenin and β-catenin expression. Survivin was reduced by γ-catenin siRNA but not β-catenin siRNA treatment. Addition of Wnt3a partially recovered the decrease of Survivin. In addition, Survivin was knocked down in primary ALL using shRNA and non-silencing shRNA control or ICG-001 (2uM) and DMSO control. Western blot analysis showed that survivin shRNA or ICG-001 treatment lead to downregulation of Survivin and γ-catenin. Using a ChIP assay we could demonstrate occupancy of TCF4 and CBP association at the Survivin promoter, which was not altered by ICG-001 in primary ALL. Moreover, ICG-001 treatment of primary ALL cells prevents CBP but not p300 occupancy. For further preclinical in vivo evaluation of ICG-001, one Philadelphia chromosome positive ALLs (Ph+) and two Ph− primary ALL were injected into sublethally irradiated NOD/SCID IL2Rγ−/-mice and treated with ICG-001 (50mg or 100mg/kg/day per subcutaneous miniosmotic pump) with or without chemotherapy including VDL for Ph− ALL (per intraperitoneal injections) or Nilotinib for Ph+ ALL (per os). For analysis we pooled the survival of all three primary leukemias. The saline control group (n=10) (MST= 55.5.days) and the ICG-001 only groups (n=3) (MST=61 days) died rapidly. The group treated with chemotherapy (n=13) had a median survival time (MST) of 85 days. In marked contrast, the group treated with the combined chemotherapy+ICG-001 (n=15) lived significantly longer (MST=100) (p<0.05). Taken together, our data shows that Survivin transcription can be mediated by γ-catenin in primary ALL and that targeting CBP/γ-catenin by using ICG-001 ALL can sensitize ALL cells to chemotherapy in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

Ki-4(scFv)–ETA′, a new recombinant anti-CD30 immunotoxin with highly specific cytotoxic activity against disseminated Hodgkin tumors in SCID mice

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3909-3914 ◽  
Author(s):  
Stefan Barth ◽  
Michael Huhn ◽  
Bärbel Matthey ◽  
Samir Tawadros ◽  
Roland Schnell ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Specific Activity ◽  
Lymphoma Cell ◽  
In Vitro Assays ◽  
Saline Control ◽  
Control Group ◽  
Immunodeficient Mice ◽  
Recombinant Immunotoxin

The human lymphocyte activation marker CD30 is highly overexpressed on Hodgkin/Reed–Sternberg cells and represents an ideal target for selective immunotherapy. We used the murine anti-CD30 hybridoma Ki-4 to construct a new recombinant immunotoxin (rIT) for possible clinical use in patients with CD30+ lymphoma. Hybridoma V genes were polymerase chain reaction-amplified, assembled, cloned, and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv obtained by selection of binding phage on the CD30-expressing Hodgkin lymphoma cell line L540cy was inserted into the bacterial expression vector pBM1.1 and fused to a deletion mutant ofPseudomonas exotoxin A (ETA′). Periplasmically expressed Ki-4(scFv)–ETA′ demonstrated specific activity against a variety of CD30+ lymphoma cells as assessed by different in vitro assays. To evaluate in vivo antitumor activity, severe combined immunodeficient mice challenged with human lymphoma cell lines were treated with the immunotoxin. The blood distribution time t½ of Ki-4(scFv)–ETA′ was 19 minutes, and its serum elimination time t½ was 193 minutes. A single intravenous injection of 40 μg rIT 1 day after tumor inoculation rendered 90% of the mice tumor free, extending the mean survival time to more than 200 days compared with 38.1 days in the phosphate-buffered saline control group (P < .001). This new rIT is a promising candidate for further clinical evaluation in patients with Hodgkin lymphoma or other CD30+malignancies.

scholarly journals Suppression of EGFR expression by antisense or small interference RNA inhibits U251 glioma cell growth in vitro and in vivo

2006 ◽  
Vol 13 (5) ◽  
pp. 530-538 ◽  
Author(s):  
C-S Kang ◽  
Z-Y Zhang ◽  
Z-F Jia ◽  
G-X Wang ◽  
M-Z Qiu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Glioma Cell ◽  
Small Interference Rna ◽  
Egfr Expression ◽  
Interference Rna

scholarly journals Klotho Alleviates Lung Injury Caused by Paraquat via Suppressing ROS/P38 MAPK-Regulated Inflammatory Responses and Apoptosis

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Zhiqiang Zhang ◽  
Qing Nian ◽  
Gang Chen ◽  
Shuqing Cui ◽  
Yuzhen Han ◽  
...  
Keyword(s):  
Lung Injury ◽  
Cell Viability ◽  
P38 Mapk ◽  
Inflammatory Responses ◽  
A549 Cells ◽  
Control Group ◽  
Ros Production ◽  
The Impact

Acute lung injury (ALI) induced by paraquat (PQ) progresses rapidly with high mortality; however, there is no effective treatment, and the specific mechanism is not well understood. The antiaging protein klotho (KL) has multiple functions and exerts significant influences on various pathophysiological processes. This work evaluated the impact of KL on PQ-induced ALI and investigated its underlying mechanisms. As for in vivo research, C57BL/6 mice were treated with PQ (30 mg/kg) intraperitoneal (IP) injection to create a toxicity model of ALI (PQ group). The mice were divided into control group, KL group, PQ group, and PQ+KL group. For in vitro experiment, A549 cells were incubated with or without KL and then treated in the presence or absence of PQ for 24 h. In vivo result indicated that KL reduced the mortality, reduced IL-1β and IL-6 in the bronchoalveolar lavage fluid (BALF), attenuated ALI, and decreased apoptosis in situ. In vitro result revealed that KL significantly improved cell viability, reduced the levels of IL-1β and IL-6 in culture supernatants, suppressed cell apoptosis, inhibited caspase-3 activation, and enhanced mitochondrial membrane potential (ΔΨm) after PQ treatment. Besides, KL effectively abated reactive oxygen species (ROS) production, improved GSH content, and lowered lipid peroxidation in PQ-exposed A549 cells. Further experiments indicated that phosphorylated JNK and P38 MAPK was increased after PQ treatment; however, KL pretreatment could significantly lower the phosphorylation of P38 MAPK. Suppression of P38 MAPK improved cell viability, alleviated inflammatory response, and reduced apoptosis-related signals; however, it had no obvious effect on the production of ROS. Treatment with N-acetylcysteine (NAC), a classic ROS scavenger, could suppress ROS production and P38 MAPK activation. These findings suggested that KL could alleviate PQ-caused ALI via inhibiting ROS/P38 MAPK signaling-regulated inflammatory responses and mitochondria-dependent apoptosis.

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